The present invention relates generally to gene therapy. More specifically, the present invention relates to somatic cell gene therapy in humans and animals.
Genetic defects in the human genome account for more than 4500 identified diseases. The resulting diseases are caused by single or multiple defects in a given gene. It is possible that many of these diseases can be alleviated, at least in part, if the deficient function can be supplied.
The concept of human gene therapy involves the introduction of a functionally active xe2x80x9creplacementxe2x80x9d gene into somatic cells of an affected subject to correct the gene defect. Retroviral vectors, because of their unique structure, modes of replication, and ability to infect a wide variety of cells, including stem cells, are ideally suited to transfer genetic material into somatic cells (Verma, 1985).
To ensure a life long supply of the replacement gene product, it is essential to introduce and express the functionally active gene in cells that proliferate during the entire adult life of the recipient. Because pluripotent stem cells in bone marrow have both self renewal capacity as well as the ability to give rise to all hematopoietic lineages, they are a popular target for the introduction of functionally active genes (Miller, et al., 1984; Williams, et al., 1984; Keller, et al., 1985; Dick, et al., 1985). Recently, hepatocytes have been used as target cells for introducing functionally active genes (Ledley, et al., 1987; Wolfe, et al., 1987).
Although the number of stem cells in adult marrow is low (0.01-0.1%), the use of high-titer retrovirus has ensured infection and gene delivery into these cells. The problem however has been that neither the foreign genes nor the retroviral vector introduced into these stem cells, the progenitor cells, or the mature end cells are efficiently expressed (Williams, et al., 1984; Joyner, et al., 1985).
Recently two groups used mouse fibroblasts to introduce and express foreign genes in mice (Selden, et al., 1987; Garver, et al., 1987b). One group implanted mice with a DNA transfected cell line and showed that the recipient mice made the gene product (growth hormone) but maintained the graft only if the mice were immunosuppressed (Selden, et al., 1987). The other group, using a chimeric retroviral vector containing the alpha1-antitrypsin gene, produced a cell line from a transduced cell and then transplanted cells from the line into the peritoneal cavity of nude mice (Garver, et al., 1987b). In both cases, cell lines were generated that would potentially be tumorigenic in mice. Neither study addresses the issue of cell maintenance in grafted mice without the use of harsh immunosuppressive agents.
In addition to the work that has been done with fibroblasts, at least one group has shown that retroviral-mediated gene transfer can be used to introduce a recombinant human growth hormone gene into cultured human keratinocytes (Morgan, et al., 1987). The transduced keratinocytes secreted biologically active growth hormone into the culture medium. When grafted as an epithelial sheet onto athymic mice, these cultured keratinocytes reconstituted an epidermis that was similar in appearance to that produced by normal cells, but from which human growth hormone could be extracted. Unfortunately, it was not possible to determine the rate of diffusion of human growth hormone from the graft site to the bloodstream. This may have been due to the fact that the surface skin graft does not efficiently vascularize.
The present invention discloses a new gene therapy method based on the use of transduced fibroblasts that are implanted in the loose connective tissue of the skin of the subject to be treated. According to the invention, transduced fibroblasts are preferably created by infecting fibroblast cells in vitro with chimeric retroviruses that contain at least one functionally active xe2x80x9creplacement genexe2x80x9d, i.e., foreign or exogenous genetic material that does not normally occur in fibroblast cells, or if it does, is not expressed by the fibroblast cells in biologically significant concentrations. Expression of xe2x80x9creplacement genexe2x80x9d can be maintained under the control of the long terminal repeat (LTR) of the retroviral vector and/or under the control of constitutive or inducible exogenous sequences. The transduced fibroblasts are then preferably fixed by culturing them in vitro in an extracellular matrix. Finally, the transduced fibroblasts are implanted subcutaneously in the loose connective tissue of the skin of the individual or animal being treated. To insure rapid vascularization of the implanted fibroblasts, an angiogenic substance such as a fibroblast growth factor is preferably placed in the loose connective tissue along with the implant. Because the fibroblasts are implanted in a highly vascularized compartment of the skin i.e., loose connective tissue of the dermis, the transduced cells, and thus their xe2x80x9creplacementxe2x80x9d gene products, have direct access to the circulatory system. As a result, the needed replacement gene products can easily and efficiently be distributed to other parts of the body. When the gene therapy is no longer needed, the implanted fibroblasts can be conveniently removed.
To overcome the prior art problem of inefficient expression, the present invention discloses an alternative strategy for somatic cell gene transfer. The new strategy uses skin fibroblasts that are infected with chimeric retrovirus containing a functionally active endogenous or foreign xe2x80x9creplacementxe2x80x9d gene. Once infected with the chimeric retrovirus, the transduced fibroblasts are preferably xe2x80x9cfixedxe2x80x9d in an extracellular collagen matrix, and then implanted in the loose connective tissue of the skin. Since this compartment of the dermis is highly vascularized, the transduced fibroblasts, and thus their xe2x80x9creplacementxe2x80x9d gene products, have direct access to the circulatory system. As a result, the needed replacement gene products can easily and efficiently be distributed to other parts of the body.
The method described herein obviates the need for established cell lines and instead uses fibroblast cells from recipient subjects. Use of a subject""s own cells minimizes the possibility of rejection. In addition, culturing the cells in an extracellular collagen matrix circumvents the problem of necrosis that would ensue following subcutaneous injection (Bell, et al., 1983). Finally, the high efficiency of retroviral infection and expression in fibroblasts (80%) essentially eliminates the need to identify transduced cells by means of selectable markers, thus greatly simplifying the overall endeavor of introduction of foreign genes.
Clinical disease states that are candidates for the gene therapy treatment method of the present invention include hemophilia, endocrine deficiency, alpha1-antitrypsin, birth control, etc.